Supplementary Information. Mitochondrial electron transport chain and biogenesis * *

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1 MitoNEET-driven alterations in adipocyte mitochondrial activity reveal a crucial adaptive process that preserves insulin sensitivity in obesity Christine M. Kusminski, William L. Holland, Kai Sun, Jiyoung Park, Stephen Spurgin, Ying Lin, G Roger Askew, Judith A. Simcox, Don A. McClain, Cai Li and Philipp E. Scherer Supplementary Information ** Adiopgenesis, and FA biosynthesis Gene expression ** Mitochondrial electron transport chain and biogenesis * * Supplemental Figure 1. Adipogenesis, FA-biosynthesis and mitochondrial pathways are upregulated in. RT-PCR confirmation of key genes obtained from microarray analyses of versus (n = 9 per group).

2 a 3 25 Lipid uptake 2 3 H-triolein uptake (arbitrary units) * b gwat mwat BAT Heart Pancreas β-oxidation Soleus Lung Liver Spleen Kidney Quad Hypoth 3 H-triolein oxidation (% of total lipid uptake) * gwat mwat BAT Heart Pancreas Soleus Lung Liver Spleen Kidney Quad Hypoth c 4 Tissue mass Tissue mass (mg per g body weight) * ** gwat mwat BAT Heart Pancreas ** Soleus Lung Liver Spleen Kidney Quad Hypoth Supplemental Figure 2. MitoNEET enhances lipid-uptake and declines the rate of β-oxidation 3 specifically in. (a) H-triolein lipid-uptake, (b) β-oxidation and (c) tissue-mass in versus tissues (2 μci/mouse in 1 ul of 5% intralipid; single tail-vein injection) (n = 6 per group).

3 a AUC anova OCR (% baseline) (X1 3 ) gwat O C R (% B as eline) Oxygen- consumption rate Olig o FC C P 2-D G AA ** gwat gwat Tim e (m in) b AUC anova ECAR (mph) E CA R (m p H per m in -1 ) Glycolytic rate Olig o FC C P 2-D G AA Tim e (m in) Supplemental Figure 3. Adipose-specific overexpression of mitoneet enhances glycolysis. Oxygen consumption rates (OCRs) (top-panel) in and whole and gwat tissue-slices (~2 mg), in addition to extracellular acidification rates (ECARs) (bottom panel) (as a measure of glycolytic rate) in and tissue-slices; in response to sequential additions of oligomycin (an ATP synthase inhibitor), FCCP (a chemical uncoupler), 2-Deoxy-D-glucose (2-DG) (an inhibitor of glycolysis) and antimycin-a (complex III inhibitor) (n = 4 per group). **P <.1; P <.1.

4 a b TRE-mitoN Χ Alb-promoter Cre Rosa26 Χ LoxP MitoNEET rtta STOP β-actin LoxP 4 Χ rtta Χ TRE- promoter mitoneet MitoNEET expression relative to β -actin Rosa26 + Dox rtta TRE- promoter mitoneet c TRE-mitoN TRE-mitoN d TRE-mitoN 25 μm 25 μm OCR (pmol -1min -1) co rb As -A tim yc cc An Su e ot R Ba sa l Supplemental Figure 4. Generation of an inducible liver-specific overexpression of mitoneet. (a) Strategy of Dox-inducible overexpression of mitoneet specifically in the liver. Liver-specific albumin (Alb)-Cre mice were crossed with the Rosa26-loxP-STOP-loxP-rtTA mice to achieve liver-specific expression of rtta. These mice were then bred with TRE-mitoN transgenic mice. Following exposure to Dox, the resulting triple transgenic mice express mitoneet only in the liver. (b) Representative Western blots and the average Dox-induced overexpression of mitoneet protein in liver tissues from and TRE-MitoN mice following Dox-HFD-feeding (6 mg/kg Dox-HFD for 3-weeks) (n = 5 per group). (c) Representative H&E stain of and TRE-MitoN livers following Dox-HFD feeding. (d) OCRs in mitochondria isolated from livers and TRE-mitoN livers (n = 5 per group).

5 a Inducible mitoneet: shrna construct targeted to the Rosa26 locus D5 NeoPA H1TetO-shRNA CAGGS-iTetR Rosa26 (RMCE exchanged) b Dox - + gwat BAT Liver - + shrnamiton c MEFs shrnamiton: Primary hepatocytes p-akt Insulin: Palmitate: MnTBAP: Total-Akt Supplemental Figure 5. Generation of an inducible constitutive knockdown of mitoneet. (a) Schematic of the Dox-inducible knockdown system of mitoneet, with the shrna construct being targeted to the Rosa26 locus. (b) Representative Western blots demonstrating the Dox-induced knockdown of mitoneet protein in, gwat, BAT and liver tissues from and shrna-miton mice following Dox-chow-feeding (6 mg/kg Dox-chow for 1 d) (+Dox) or standard chow-feeding (-Dox) (n = 4 per group). (c) Representative Western blots showing insulin signaling (phospho-akt (Ser473) (p-akt) and total-akt) in Dox-treated (1 mg/ml) and shrna-miton MEFs (left panel), in addition to primary hepatocytes (right panel) derived from mice and shrna-miton mice. Expression levels were examined in response to treatments with, or without insulin, palmitate and the anti-oxidant, MnTBAP (n = 4 per group).

6 Supplemental Table 1. A summary of the differentially regulated pathways and genes identified by microarray cluster analyses from and. Gene abbreviations, definitions, in addition to fold-alterations and significant differences between and groups are indicated (n = 9 per group). *P <.5; **P <.1. Pathway Gene Gene def inition Fold-change P-value Adipogenic and lipogenic transcription factors: Pparγ Peroxisome prolif erator activated receptor γ C/ebpα/β CCAAT/enhancer-binding protein α/β Srebp1c Sterol regulatory element binding transcription f actor 1 Lxrα Nuclear receptor subf amily 1, group H, member 3 Klf15 Kruppel-like f actor 15 Adipoq Adiponectin TG synthesis, NEFA re-esterification and fatty acid biosynthesis: Lpin1 Pepck-C Dgat1/2 Glut4 Gnpat Fasn Fads1/2 Mcat Lipin 1 Phosphoenolpyruvate carboxykinase, soluble Diacylglycerol O-acyltransf erase 1 Solute carrier family 2 (facilitated glucose transporter) member 4 Glyceronephosphate O-acyltransf erase Fatty acid synthase Fatty acid desaturase 1/2 Malonyl CoA:ACP acyltransf erase Lipid-droplet associated proteins: Plin1 Perilipin 1 Fsp27 Cell death-inducing DFFA-like effector c Fatty acid uptake, transport and oxidation: Fabp5 Fatty acid binding protein 5 Fatp4 Fatty acid transporter protein 4 Cd36 Cd36 antigen Cpt1 Carnitine palmitoyl transf erase I PPARα Peroxisome prolif erator activated receptor-α / / / **.458*/.382*.5.377*.429*.187*.153*.367*.58/.342*.138*.343*.332*.42*/.443*.142*.374*.291*.297*.138*.42*.57** N.S Beta-adrenergic signaling: Adrβ1/2/3 Adrenergic receptor β 1/2/3 1.18/1.9/1.36 N.S/N.S/.16*

7 Supplemental Table 2. A list of RT-PCR primer sequences of differentially expressed genes identified by the microarray cluster analysis from derived from mice versus MitoN- Tg mice.

8 Supplemental Table 3. Ad libitum chow-fed and fasted (24 h) body-weights and serum parameters in mice versus mice (n = 7 per group). Fed Fasted (24 h) Body-weight (g) Glucose (mg/dl) Insulin (ng/ml) Triglyceride (mg/dl) FFA (mmol/l) Glycerol (mmol/l) Adiponectin (μg/ml) 28.7 ± ± ± ± ±.2.15 ± ± ± ± ± ± 8.2**.12 ±.2.16 ± ± ± ± ± ± ±.3.19 ± ± ± ± ± ± ±.4*.26 ±.2** 21.7 ±.1 Data obtained from 12-week old male FVB mice. * P <.5; ** P <.1; P <.1. Supplemental Table 4. Chow-fed ob/ob mice versus ob/ob mice fasted (3 h) bodyweights and serum parameters (n = 8 per group).

9 Supplementary Methods Animals. Generation of an inducible shrna-miton knockdown mouse model was developed as described by Seibler et al. 1 (Taconic Artemis). Briefly, the inducible shrna transgene was constructed by introduction DNA encoding an shrna of the sequence (5'- GGCCTTGCTACTGAAACATTTCAAGAGAATGTTTCAGTAGCAAGGCC-3') into a cassette consisting of a truncated Neomycin selectable marker, an H1-tetO promoter/operator for shrna expression and a CAGGS-iTetR gene (for Dox-dependent repression of shrna expression) all flanked by incompatible FRT sites for subsequent Flpe-mediated RMCE (Recombinase Mediated Cassette Exchange) 2 in ES cells. The 5' underlined 19 nucleotides are sense and the 3' underlined nucleotides are antisense to mitoneet transcripts. The shrnamiton transgene cassette was targeted to the RMCE compatible Rosa 26 locus in ES cells (strain 129S6). Six candidate shrnas were tested in this transgenic format for optimal inducible knockdown of mitoneet RNA in targeted ES cells. Targeted ES cells expressing the most potent shrna molecule were utilized for generation of mice by blastocyst injection. Mice used in the present study have been backcrossed to C57/BL6 background. To generate a Doxinducible mitoneet overexpression mouse model, mitoneet cdna and a Kozak sequence of GCCGCCACC were engineered into the ptre-tight vector (Clontech) with Xba I sites. The expression of mitoneet is controlled by 7 tandem repeats of tetracycline responsive elements in front of a minimum CMV promoter in the ptre-tight construct. A rabbit β-globin 3 UTR was included to stabilize the transcript and enhance the translation. After Nae I, and ApaL I digestion and purification, ptre-mitoneet DNA was injected to embryos into a pure C57/BL6 background by the transgenic core facility at UTSW. The liver specific albumin-cre transgenic mice and the Rosa26-loxP-STOP-loxP-rtTA transgenic mice were purchased from the Jackson

10 Laboratories. Albumin-Cre mice were firstly bred with Rosa26-loxP-STOP-loxP-rtTA mice to obtain mice homozygous for both transgenes. To achieve inducible expression of mitoneet, TRE-mitoNEET mice were then crossed with double homozygous albumin-cre and Rosa26- loxp-stop-loxp-rtta mice. The resulting triple transgenic animals were used for experiments. Age-matched transgenic animals with albumin-cre and Rosa26-loxP-STOP-loxP-rtTA genotype, but lacking the TRE-mitoNEET transgene were used as controls. Transmission electron microscope (TEM). For TEM analysis, fresh tissues were fixed by perfusion with 4% paraformaldehyde and 1% glutaraldehyde in.1 M sodium cacodylate buffer. Fixed tissues were then transferred to 2.5% glutaraldehyde in.1 M sodium cacodylate buffer, post-fixed in buffered 1% osmium tetroxide, en bloc stained in 4% uranyl acetate in 5% ethanol, dehydrated with a graded series of ethanol, then embedded in EMbed-812 resin. Thin sections were cut on a Leica Ultracut UCT ultramicrotome and post-stained with 2% uranyl acetate and lead citrate. Images were acquired on a FEI Tecnai G 2 Spirit TEM equipped with a LaB 6 source and operating at 12 kv. Primary hepatocyte isolation and treatment. Primary hepatocytes were isolated from 12-week old male C57/BL6 and shrna-miton mice. Following overnight plating, hepatocytes were treated with either palmitate (4 μm) or MnTBAP (1 mg ml -1 ) for 4 h, then acutely with insulin (16 mm) for 1 min. Protein was extracted for Western blot analysis. Mitochondrial membrane potential (ΔΨm). For ΔΨm, MEFs and shrna-miton MEFs (1x1 5 ) were incubated with or without Dox (1 μg ml -1 ; 48 h). Cells were treated with 5 μm TMRM for 2 min at 37 C, with an additional well containing control FCCP (2 μm for 3 min at 37 C). All images were obtained using a confocal microscope (Leica TCS SP5) at 63

11 magnification. In parallel, for stable transfection, the pcb7-mitoneet construct was transfected into 3T3-L1 preadipocytes using Lipofectamine 2 (Invitrogen). The transfected cells were selected by hygromycin B (125 μg ml -1 ), then individual cell clones were isolated, expanded and analyzed for expression levels of recombinant mitoneet protein by Western blot. Two clonal lines, with low- and high-mitoneet expression levels respectively, were utilized for ΔΨm analysis, along with 3T3-L1 preadipocytes pre-treated for 12 h with 1. μm TZD (rosiglitazone) or control vehicle. ΔΨm was measured using 3,3 -dihexyloxacarbocyanine iodide (DiOC 6 ; Sigma) staining. Briefly, cultured cells were washed with PBS, then incubated for 15 min at 37 C in 1 ml of serum-free culture medium containing 5 nm DiOC 6. Following centrifugation, cells were re-suspended in PBS, then immediately analyzed by flow cytometry on a FACscan (Becton Dickinson Immunocytometry Systems). References 1. Seibler, J., et al. Reversible gene knockdown in mice using a tight, inducible shrna expression system. Nucleic Acids Res 35, e54 (27). 2. Baer, A. & Bode, J. Coping with kinetic and thermodynamic barriers: RMCE, an efficient strategy for the targeted integration of transgenes. Curr Opin Biotechnol 12, (21).